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RiboPure Yeast Kit Instructions
Pdf of kit protocol, for using AMBION RiboPure Yeast, P/N 1926: http://tools.invitrogen.com/content/sfs/manuals/1926ME.pdf Before starting: '''preheat Elution Solution to 95ºC Check wash soliution 1 and the 10%SDS for precipitation and heat to 37ºC if precipitate exists inspect filter cartridges and gently push the filter down to the bottom of the cartridge if needed. '''Protocol: 1. Collect cells (up to 3x10^8) from a yeast overnight, you probably want log phase culture ~1.5mL, by centrifuging for 2mins at >12,000x g (i.e. top speed on a Microfuge). 2.'''Resuspend cells in lysis reagents by adding (in order): A) 480uL of lysis buffer; B) 48uL of 10% SDS; C) 480uL of Phenol:Chloroform:IAA . All three of these reagents are part of the kit, and additional RNAse free Phenol:Chloroform:Isoamyl Alcohol is in the door of 4C #3 if you need it. '''3.Vortex vigorously for 10-15sec. 4. '''For each yeast sample, pour 750uL of ice-cold Zirconia Beads (part of kit) into an autoclaved 1.5ml screw cap tube. * Estimate the volume of beads by pouring them to a height of about 2.5cm (1inch) from the bottom of the tube. See pic on right. '''5. Transfer the mixture of cells and lysis reagent to one of the prepared tubes containing 750uL of cold Zirconia Beads and securely fasten the lid. 6. Place sample tubes in the vortex adapter on Mark's microfuge , and vortex on about 7 for 10 minutes. If the vortexer is scooting around the bench, turn the speed down slightly. 7. Centrifuge the sample tubes for 5min at 16,100x g (top speed on Microfuge) at room temperature to separate the aqueous phase, containing the RNA, from the organic phase. 8. '''Transfer the aqueous (top) layer, which contains the partially purified RNA, to new, labeled 15ml Conical Tubes . '''9. Add 1.9mL of Binding Buffer (from kit) and 1.25mL of 95% EtOH and mix thoroughly. 10. Draw samples through cartridges (use manifold method; for alternative centrifuge method see pdf link at top). Manifold: ''' '''11. Add labeled Syringes to vacuum manifold by cutting off the luer attachment. (syringes P/N: BD 309646, manifold Qiagen P/N 19413). 12. Put a Filter Cartridge in each syringe. 13. '''Fill each Filter Cartridge with 800uL of RNA solutions from 15ml conicals . Vacuum the solutions through the filters. '''14. '''Repeat step 13 until all of your RNA solution has passed through the filter. '''15. Add 700uL Wash Solution 1 to the filter cartridge and draw through the filter. 16. '''Wash the filter by adding 500uL Wash Solution 2/3 and draw through the filter. '''17. '''Repeat step 16. '''FINISHED WITH MANIFOLD HERE 18. Centrifuge the filter cartridge for 1min to remove excess wash Make sure to use the centrifuge for this step, not the manifold. Discard flow through. 19. '''Get a new flip top tube for each filter cartridge. '''20. Add 50uL of the heated Elution Solution (95*C) to the center of each cartridge. 21. '''Centrifuge for 1min. '''22. Add an additional 50uL of the heated Elution Solution to the center of each cartridge 23. Centrifuge for 1min; keeping the eluate (flow-through)! 24. '''Remove and discard column, keeping the solution in the flip top tube. '''25. Add the following to each flip top tube: A)10uL 10X DNase 1 Buffer; B) 4uL DNase I; and then incubate at 37º for 30 minutes. 26. Resuspend DNase Inactivation Reagent by vortexing it. 27. Add 11uL of DNase Iaction Reagent to each tube. 28. '''Mix by vortexing it and allow it to sit for 5 minutes at room temperature. '''29. Centrifuge the tubes at top speed for 3 minutes. 30. '''Collect the supernatant and place it in a RNase free screw cap tube . Store at -80C. AFTER: you should spec 2ul of your RNA on the nanodrop to determine concentration (ng/ul), set nanodrop to RNA, and write this concentration on the side of the tube. You should also run an RNA GEL to determine quality- for this use TBE rather than TAE for gel and buffer, and run WITHOUT EtBr, staining with EtBR in TBE for 45 mins and destaining with TBE no EtBr (or milliQ h20) for 10 minutes. See below for RNA GEL results. '''AMBION RiboPure Yeast, P/N 1926 5 ml syringes BD P/N 309646 QiaVac 24 plus Qiagen P/N 19413. ' The plastic drain plug of the QiaVac 24 will strip almost immediately; replace it with McMaster-Carr part 93190A212, 316 stainless steel bolt fully threaded 5/8" X 18 tpi.' Additional RNAse free 1.5ml eppie tubes: Axygen / Genesee Scientific P/N 22-284